Characterization of the molecular basis for virus attachment to cells has importance both for understanding virus tropism and for developing agents that inhibit virus binding or alter the specificity of binding. Recently, a cellular receptor for adenovirus type 2 and other closely related serotypes was identified. This receptor, encoded by a single gene on human chromosome 21 (Mayr et al., J. Virol. 71: 412-8 (1997)), is a 46 kD glycoprotein which also serves as a receptor for group B coxsackieviruses (CBV) and thus was termed CAR. CAR mRNA is present in many human tissues. A broad tissue distribution of CAR protein expression correlates with the broad tropism of CBV, but subgroup C adenoviruses that are known to bind CAR have a much more restricted tropism limited. primarily to the upper respiratory tract. Thus, other factors in addition to receptor availability clearly have important roles in determining adenovirus tropism. Although adenovirus binds to CAR with high affinity (Mayr et al., J. Virol. 71: 412-8 (1997); Wickham et al., Cell. 73: 309-19 (1993)), virus titers are significantly reduced on cells with down-regulated CAR expression (Freimuth, P., J. Virol. 70: 4081-5 (1996)). These results suggest that adenovirus infection in vivo may be restricted to cells which express CAR at levels above a minimum threshold concentration. CAR protein levels are relatively low on the apical surface of differentiated (ciliated) respiratory epithelial cell cultures, which may account for the poor efficiency of adenoviral gene transfer to human lung tissue in vivo.
Adenovirus binding to CAR results from an interaction between rod-shaped proteins located at the capsid vertices, called viral fibers, and the extracellular region of CAR. The monomers of this homotrimeric fiber protein range in size from 30 to 65 kDa depending on the serotype (Huang et al., J. Virol. 73: 2798-2802 (1999)). They are composed of a conserved amino terminal tail that mediates their interaction with the Ad penton base, a variable-length elongated (shaft) domain, and a carboxyl-terminal globular domain, termed the knob, which mediates the high-affinity interaction with its cellular receptor. The knob domain of adenovirus type 5 (Ad5) was expressed in E. coli as a soluble, trimeric, biologically active protein, and its 3-dimensional structure was determined by x-ray crystallography (Xia et al., Structure 2: 1259-70 (1994)).
The predicted amino acid sequence of CAR suggests a structure consisting of two extracellular domains related to the immunoglobulin IgV and IgC2 domain folds (Bork et al., J. Mol Biol. 242: 309-20 (1994); Bergelson et al., Science 275: 1320-3 (1997); Tomko et al., Proc. Natl. Acad. Sci. USA 94: 3352-6 (1997)), a single membrane-spanning region, and one carboxy-terminal cytoplasmic domain. Regions of CAR necessary for binding the fiber knob domain have not yet been determined.
The present invention relates to a mutant adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain, the viral particle encoded by the genome being characterized by a significantly weakened binding affinity for CARD1 relative to wild-type adenovirus. Preferably, the mutant adenovirus is adenovirus serotype 2 or serotype 5. The mutation may be in sequences which encode the AB loop of the fiber protein knob domain. Specific residues and mutations are described. Alternatively, the mutations which cause significantly weakened binding affinity for CARD1 may be in sequences which encode the HI loop of the fiber protein knob domain of the encoded viral particle. Specific residues and mutations are described.
Another aspect of the present invention is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type. This method is performed on adenoviruses which bind CARD1. Residues of the adenovirus fiber protein knob domain of the adenovirus, which are predicted to alter D1 binding when mutated, are identified from the crystal structure coordinates of the AD12knob:CAR-D1 complex. A mutation which alters one or more of the identified residues is introduced into the genome of the adenovirus, and whether or not the mutant produced exhibits altered adenovirus-CAR binding properties is determined. This method can be used to produce a mutant adenovirus which, under physiological conditions, has significantly weakened binding affinity for CARD1 relative to wild type adenovirus or which binds a receptor other than CARD1, including an engineered receptor. The introduced mutation may result in an amino acid substitution, an amino acid deletion, or an amino acid insertion in the encoded viral particle. Introduced mutations may serve to alter the conformation of one or more residues of knob which participate directly in D1 binding. Such residues include residues of the AB loop, the CD loop, the DE loop, the FG loop, the E strand and the F strand. Alternatively, the mutation may be introduced in a codon encoding the residue of knob which participates directly in D1 binding. Specific residues in the AB loop, the CD loop, the FG loop, the E strand, the F strand, and the DE loop which participate directly in binding are identified.
Another aspect of the present invention is a method for identifying an inhibitor of adenovirus binding to CAR. In the method, a three-dimensional structure derived by X-ray diffraction from a crystal of adenovirus knob trimer bound to CARD1 is provided and then employed to design or select a potential inhibitor. The potential inhibitor is synthesized and then whether or not the potential inhibitor inhibits adenovirus binding to CAR is determined. The crystal of the Ad12knob:CARD1 complex which is used in the method preferably has P4332 space group symmetry with a cubic unit cell with 167.85 angstroms per side. Atomic coordinates are preferably obtained by means of computational analysis. A set of atomic coordinates which define the three dimensional structure are provided. In one embodiment, the potential inhibitor is designed to interact non-covalently with one or more residues of the adenovirus fiber knob protein domain. In another embodiment, the potential inhibitor is designed to interact non-covalently with one or more residues of CARD1. Specific residues for covalent and non-covalent interaction are listed. In another embodiment, the potential inhibitor is designed to interact non-covalently with residues which line a cavity formed during adenovirus knob trimer/CARD1 binding. The potential inhibitor can be designed by identifying chemical entities or fragments capable of associating with the adenovirus knob trimer, and assembling the identified chemical entities or fragments into a single molecule to provide the structure of said potential inhibitor. Such an inhibitor may be designed de novo or from a known inhibitor. Methods of inhibition include competitive inhibition, non-competitive inhibition and uncompetitive inhibition.